Metalloproteases (MMPs) play a pivotal role in tissue remodeling during morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Pericellular proteolysis catalyzed by MMPs is an important factor in defining the microenvironment of the resident cells of normal and neoplastic tissues. Malignant cells exploit MMPs to promote tumor invasion and metastasis. Since the cell surface MMP-2 activation complex ((MT1-MMP)2/TIMP-2/MMP-2) was described in our lab in the 1990s, we have studied molecular mechanisms of the spatial regulation catalyzed by this complex to elucidate the role of MMPs in cell - ECM interactions. We have recently identified some remarkable features of MMPs. We showed that i. MMPs interact with collagen via a substrate surface diffusion mechanism. Both trans-membrane MT1-MMP and secreted MMP-1, -2 and 9 can diffuse on the surface of native collagen fibrils;ii. MMP-1 acts as a unique, diffusion-based, ATP- independent motor enzyme driven by collagen proteolysis;iii. The extra-cellular portion of MT1-MMP interacts with collagen through a similar biased-diffusion mechanism;iv. Complex formation of MMP-2 C-terminal domain with the inhibitor TIMP-2 does not affect the rate of diffusion. Thus the entire membrane tethered collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 is mobile relative to the underlying collagen substratum. These findings have profound implications for a mechanistic understanding of the role of MMPs in cell locomotion in a collagen rich microenvironment. We thus propose a model for a Mobile Cell Surface Collagenolytic Interface. In this model we hypothesize that the membrane tethered Collagenolytic Complex assists cell locomotion by virtue of its ability to slide directionally along the underlying collagen fibril. This mechanism is likely to be instrumental in orchestration of cell adhesion-desorption events during cell locomotion in collaboration with the cytoskeleton-integrin adhesion apparatus. To support this notion we now present evidence that the activity of the cell surface collagenolytic complex aids in force generation that cells exert in 3D collagen tissue constructs. This motivates us to further examine whether pericellular collagenolysis contributes to force generation by cells in a collagen- rich microenvironment. Thus here we propose i. To complete the investigation of the MT1-MMP as a proteolysis driven Brownian ratchet;ii. To define the structure-function relationship in MMP-1 and/or MT1-MMP relevant to the mechanism of substrate surface diffusion and iii. To determine the contribution of pericellular collagenolysis to force generation by cells in a collagen rich microenvironment using several experimental approaches including traction force microscopy in 2D cultures, the force measurements in 3D tissue constructs, and finally to measure the force that cells can exert on an individual collagen fibril utilizing cells with ablated enzyme gene(s) and collagen with mutated collagenase cleavage site. These studies will provide the necessary background for further investigation of how cells utilize pericellular proteolysis to orchestrate the adhesion-detachment events in cell locomotion and tissue remodeling that is a key process in metastatic tumor invasion. Public Health Relevance: Matrix Metalloproteases (MMPs) are the specialized group of enzyme with unique ability to catalyze turnover of extracellular matrix components such as collagen. These enzymes play a pivotal role in tissue remodeling during normal processes of morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Malignant cells exploit MMPs to promote tumor invasion and metastasis. The goal of this proposal is to provide for a better understanding of the molecular mechanisms of spatially regulated peri-cellular proteolysis catalyzed by MMPs and its functional role in numerous normal and pathological conditions.